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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diisopropylamine
- Physical state: water-like liquid
- Analytical purity: 99.99%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: NBP 2853706
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: Males- 279.8 to 357.0 grams. Females- 181.0 to 211.4 grams
- Fasting period before study: no
- Housing: Individual suspended stainless steel cages, over paper bedding
- Diet: Ralston Purina RODENT CHOW No. 5002, ad libitum
- Water: ad libitum (St. Louis public water sup
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-74
- Humidity (%): 35-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test substance was applied daily for five days to an approximately 25 cm² shaved area of the upper back with a microliter syringe or a micropipet. The area of exposure was left unoccluded. A plastic collar was applied during the 6 hour exposure. The skin was not wiped clean of any remaining test substance after each exposure. Volme applied was based on individual animal’s body weight. Controls were treated identically, except that nothing was applied to the skin.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography using a flame ionization detector Test Material Stability: Analysis of samples placed on watch glass for 6 hours at 100 degrees F, or at ambient temperature (closed container, 56 days)
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 50 and 150 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Diisopropylamine was applied to and left unoccluded on the shaved skin (approximately 25 cm²; approximately 10% of the total body surface area) of groups of 3 male and 3 female Sprague-Dawley rats at targeted doses of 0, 50, 150, 450, 1350 or 2000 milligrams per kilogram (mg/kg) per day, for five consecutive days. Plastic collars were used to prevent ingestion of the test material. Negative controls were handled identically to treated animals, except nothing was applied to the skin. The animals were weighed pretest, and were weighed and sacrificed on the day following the last exposure. Necropsies were not performed.
All 3 males and females at the 2000 mg/kg/day level were sacrificed after receiving three applications of the test material, due to extreme irritative effects to their skin. Body weight losses occurred in all animals at the 1350 mg/kg/day level. Clinical signs of mild to severe skin discoloration, dryness, redness, scabs and/or bleeding were seen in the majority of animals at the 450 and 1350 mg/kg/day levels.
Based on the above results, dose levels of 0, 15, 50 and 150 mg/kg/day were selected for the one month study.

- Post-exposure recovery period in satellite groups: none

Examinations

Observations and examinations performed and frequency:
In-life Observations:
Checks for Mortality and Moribundity: Twice daily
Detailed Obsenations for Signs of Toxicity: Once weekly
Body Weight and Food Consumption Measurement: Once weekly

Clinical Pathology:
Frequency: Once; at termination of study
Approximate Number of Animals: 10/level/sex
Fasting: Food withheld overnight prior to blood collection
Collection Site: Posterior vena cava of anesthetized animals

Hematology Determinations:
Total erythrocyte count (RBC), total leukocyte count (WBC), platelet count (PLT), hematocrit (HCT), level of hemoglobin (HGB), and red blood cell indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)] : Whole blood treated with anticoagulant (EDTA pretreated commercial tubes) processed on a COULTER S Plus II blood cell counter using the manufacturers’ methods.
Leukocyte differential: Thin blood smears on labelled glass slides prepared$ stained with Wright’s stain, and examined microscopically
Reticulocyte count: A portion of the EDTA sample was mixed with a vital stain (methylene blue), a slide was prepared and examined microscopically

Blood Chemistry Determinations:
Albumin, total protein, blood urea nitrogen (BUN), total bilirubin, direct bilirubin, glucose, glutamic pyruvic transaminase (D-SPT/ALT), alkaline phosphatase, glutamic oxaloacetate transaminase (D-SOT/AST), gamma glutamyl transpeptidase (gamma-GT), globulin, creatinine, cholesterol (Chol), calcium, phosphate, chloride, sodium, and potassium: Serum harvested by centrifugation of samples submitted in commercial clot tubes assayed with a KDA clinical analyzer using the manufacturer’s methods. Globulin determined by subtraction of albumin value from total protein value.
Sacrifice and pathology:
- Gross Pathology
Scheduled Sacrifices:
Frequency: At study termination (approximately 4 weeks)
Number of Animals: All survivors
Organs weighed: Brain, heart, kidneys, liver, spleen, testes with epididymides
Extent of Examination: External and internal. Internal cavities opened and organs examined in situ and then removed. Hollow organs opened and examined.
Organs Retained (when present): aorta, adrenals, bone with marrow, brain, caecum, esophagus, eyes, heart, kidneys, colon, liver, lung (with mainstem bronchi), lymph node (mesenteric and e bow), mammary gland, muscle (quadriceps femoris), nasal passages, sciatic nene, pancreas, prostate, pituitary, submaxillary salivary gland, skin (treated and untreated areas), small intestine, spinal cord, spleen, stomach, testes with epididymides, thymus,
thyroid/parathyroid, trachea, uterus (corpus and cervix) , ovaries, urinary bladder
Fixative: Eyes- buffered 0.5% glutaraldehyde, 5% formalin
All other tissues- 10% neutral buffered formalin

- Histopathology
Tissues Examined: All retained tissues from all animals at control and highest dose level, target tissues at middle and low levels
Tissue Preparation: Fixed tissues washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin
Examination: Light microscopy
Statistics:
The following statistical procedures were used to detect statistically significant differences between treated animals and their respective controls:
Dunnett’s Multiple Comparison Test (two-tailed)): body weights, food consumption, non categorical clinical pathology data, absolute organ weights
Mann-Whitney Test with Bonferroni Inequality Procedure: Organ weight/body weight ratios
Fisher’s Exact Test with Bonferroni Inequality Procedure: Incidence of microscopic lesions
Other statistical routines used for some data were: Bartlett’s Test to evaluate homogeneity of variances, Analysis ofVariance to determine if the sample (group) means could be considered as an estimate of a common population, and Grubb’s Test to detect outliers.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities during this study.
Abnormalities of the skin at the site of application were the only clinical signs considered to be a result of testing. These consisted primarily of mild dryness and scab formation, and were most prevalent in high level females (5 of 10 animals). There was not a clear dose-response relationship for either effect, and they were probably merely the result of the repeated of a volatile agent (dryness), or the shaving of the site (scab formation).

BODY WEIGHT AND WEIGHT GAIN
Males at all treated dose levels failed to gain as much body weight as controls, although statistical significance was never achieved; all male test levels were approximately 7.5% lighter in group mean body weight than controls at the end of testing.
Females at all test levels had weight gains comparable to their respective controls.

FOOD CONSUMPTION
Food consumption for test groups of both sexes was comparable to or greater than that of controls, both on a grams/day (GM/DAY) and grams/kilogram of body weight/day (GM/KG DAY) basis throughout most of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY
All changes in hematologic and serum biochemical parameters were of small magnitude, not dose-related, were in the opposite direction of that expected from toxic injury, and/or were within established ranges of normal for rats of this strain and age. Therefore, changes in these parameters were considered to be biologically insignificant and/or unrelated to treatment.

ORGAN WEIGHTS, GROSS PATHOLOGY AND HISTOPATHOLOGY
There were no changes in relative or absolute organ weights that were considered to have been related to treatment. Decreases in absolute heart and liver weights were seen in all levels of treated males, and decreased relative heart weight was noted in high level males as well as decreased absolute brain weight in high level females. Also at the two highest treatment levels, relative testicular weights were increased. Mild splenic congestion was present in 2/10, 1/10 and 2/10 males and 4/10, 1/10 and 6/10 females from low, intermediate and high dosage groups, respectively. There were no other gross or microscopic changes considered related to treatment.

Effect levels

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Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 150 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Any obvious adverse effects were observed
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
>= 150 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Any obvious adverse effects were observed
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
2.19 mg/cm² per day
Sex:
male
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
1.24 mg/cm² per day
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analyses of the test material stability indicated that the neat test material was stable at room temperature over a length of time exceeding that of the study. All of the test material volatilized after 30 minutes of simulated in-use conditions.

Applicant's summary and conclusion

Conclusions:
dermal NOAEL in rats >= 150mg/kg (4 weeks study)
Executive summary:

The Repeated dermal toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats according to a protocol similar to OECD N°410 guideline. Groups of 10 male and 10 female Charles River CD(SD)BR rats received an open dermal exposure to 15, 50, and 150 mg/kg of undiluted Diisopropylamine five times a week for 1 month. The animals were monitored forsigns of toxicity and changes in hematology and clinical chemistry parameters. Necropsy was performed on all rats. A concurrent untreated control group of animals was also monitored.

Body weight gain and feed consumption in the test groups were comparable to those of the control group. Mild skin dryness was observed at the sites of application. Because this manifestation did not appear to be dose related, the authors attributed it to the repeated applications and evaporation rather than a compound related effect. No treatment-related changes were found among the hematology and clinical chemistry parameters investigated. Decreases were found inthe absolute and relative heart weights of the male rats from the high-dose group, andincreases in the absolute and relative testes weights of the males from the mid- andhigh-dose groups. However, the authors noted that no microscopic changes were found in these organs and that mild splenic congestion was present in all groups. No dose-response relationship or concomitant changes in splenic weights and hematology parameters were seen; thus, the splenic congestion was not considered related to the treatment.

The authors concluded that there was no evidence of dermal toxicity in rats treated with doses up to 150 mg/kg/day Diisopropylamine for 1 month.